The dismal prognosis of patients diagnosed with invasive and metastatic cancer points to our limited arsenal of effective anti-cancer therapies. The central importance of EGFR/HER2/RAS pathway activation has been well established in neoplastic transformation and tumorigenesis. An important goal in cancer biology is to identify means of countervailing activated EGFR/HER2/RAS signals and reverse malignant transformation. Drosophila SEVEN-IN-ABSENTIA (SINA) and its human homolog SIAHs belong to an evolutionarily conserved family of the RING domain E3 ligases that are an essential downstream signaling component required for RAS signal transduction. We have made three observations that form the basis for this application: (1) we show that SIAH is a novel biomarker in human cancer; (2) we demonstrate and that proper SIAH function is critical for RAS signaling pathway. By inhibiting SIAH2 function, we have completely blocked tumorigenesis in eleven of the most aggressive human cancer cell lines known. (3) By using anti-SIAH2 molecules, we have also completely inhibited metastasis (extravasation) in four of the most aggressive and metastatic human cancer cell lines in nude mice. Certainly, we are cognizant that the results are still preliminary due to the imperfections of the xenograft models. To address the imperfections and expand these observations, we propose to accomplish the following two aims in this revised grant proposal: (I) To delineate SIAH2 function in cancer, through a proteomic approach, we identified three LIM domain proteins, TRIP6/FHL2/LPXN, as novel SIAH2-interacting proteins from three of the most aggressive human cancer cell lines used. We provide extensive biochemical evidence to show that TRIP6/FHL2/LPXN are bona fide SIAH2 substrates. These new SIAH2 substrates can successfully rescue cell motility and viability defects observed in SIAH2-deficient cancer cells, suggesting that these focal adhesion proteins may represent one of many diverse signaling modules that function downstream of SIAH2 in mediating aspects of RAS-dependent signaling in normal development and cancer. This finding may provide a novel mechanism to explain how ERBB/RAS activation reduces cell adhesion, increases cell motility and promotes invasion and metastasis in tumor cells. (II) In Aim 2, we will extend the observations made in nude mice to a more robust in vivo system to test the anti-tumor efficacy of anti-SIAH molecules in transgenic mouse cancer models. These preclinical studies are necessary to demonstrate the effectiveness of anti-SIAH2-based anticancer strategy in animal models. Our preliminary data indicate that SIAH2-insufficiency blocks K-RAS-mediated lung tumor formation in mice. We will validate the molecular regulation of SIAH2- TRIP6/FHL2/LPXN interaction using the inducible mouse models to understand the molecular dynamics of SIAH2 action in modulating cell junction, adhesion and migration in response to RAS activation in mice. Ultimately, we hope that the knowledge gained from these studies will be useful to identify novel anti-SIAH2- based molecular mechanism(s) to inhibit tumor growth and metastasis in the future.